A protein specifically expressed in the prostate has been identified and characterized as the prostate-specific antigen (PSA) (Hara, M. and Kimura, H. (1989) Two prostate-specific antigens, .gamma.-seminoprotein and .beta.-microseminoprotein, Lab. Clin. Med. 113:541-548; Wang, M. C. et al. (1979) Purification of a human prostate specific antigen, Investigative Urology 17:159-163; Chan, D. W. et al. (1987) Prostate-specific antigen as a marker for prostatic cancer: a monoclonal and a polyclonal immunoassay compared, Clinical Chemistry 33:1916-1920; Emtage, L. A. et al. (1987) The role of prostate specific antigen in the baseline assessment of patients undergoing hormone therapy for advanced prostate cancer, British Journal of Urology 60:572-577.)
The tissue specific expression of this protein implies the existence of a unique mechanism by which the PSA gene can be transcribed only in prostate cells. The 5', .apprxeq.620 bp, flanking sequence of the PSA gene was studied and identified as the promoter for the PSA gene (Riegman, P. H. et al. (1991). The promoter of the prostate-specific antigen gene contains a functional androgen responsive element, Molecular Endocrinology 5:1921-1930). Using electroporation methods to transfect LNCaP prostate cell line, the PSA promoter isolated from a prostate carcinoma patient (PCPSA-P) has shown strong tissue specificity and responsiveness to androgen stimulation (Pang S, et al. (1995). Prostate tissue specificity of the prostate-specific antigen (PSA) promoter isolated from a patient with prostate cancer, Human Gene Therapy 6:1417-1426).
The very high expression of the PSA gene in some patients (more than 1000-fold increase in some cases) with advanced prostate cancer suggests that the regulation of PSA gene expression may involve other DNA sequences besides the PSA promoter. In other words, the transcription of PSA gene may be synergistically controlled by two regulatory elements. Similar cases have been reported for other genes.
A tissue-specific distal promoter for the human skeletal alpha-actin gene has been found in myogenic cells, and deletion of this distal promoter results in a ten-fold reduction in transcription (Muscat, G. E.; Perry, S., Prentice, H.; Kedes, L.; The human skeletal alpha-actin gene is regulated by a muscle-specific enhancer that binds three nuclear factors, Gene Expression 2:111-126). It is possible that PSA gene expression is controlled in a similar manner.
Addition of a distal PSA promoter or positive regulatory sequence should likely increase promoter activity without compromising its tissue specificity. Since gene expression is usually controlled by both promoter and enhancer, there may also be a prostate tissue-specific enhancer in the flanking regions of the PSA gene.
Using polymerase chain reaction (PCR) cloning methods, a DNA sequence upstream of the PSA promoter was amplified and obtained. The resulting DNA fragment was cloned into a plasmid containing the PSA promoter and luciferase gene. The resulting construct was tested using both lipofection and electroporation to introduce them into PSA-producing prostate cell line LNCaP and control cell lines including non-PSA-producing cell line PC-3 and DU145, renal cell line R11 and breast tumor cell line MCF-7.
Deletion tests were performed in order to define the essential region of the regulatory element. A potent tissue-specific DNA fragment of approximately 820 bp was identified, called the prostate specific antigen promoter (PSAP; Riegman, P. H. J. et al. (1991). The promoter of the prostate-specific antigen gene contains a functional androgen responsive element, Mol. Endocrinol. 5(12):1921-1930). PSAP has been identified, characterized and found to be a tissue specific promoter regulating PSA gene expression.
The discovery herein involves identifying additional regulatory sequences. Sequences upstream of the PSA promoter were screened and a DNA fragment of 822 base pairs (bp) positively regulating gene expression was found in PSA-producing prostate cells. This 822 bp DNA fragment is a newly identified PSA gene regulatory sequence (PSAR). PSAR in combination with our previously identified PSA promoter (PCPSA-P) exhibited maximal activity using DNA transfection by electroporation in PSA-producing LNCaP cell line.
With the addition of 10 to 100 nM dihydrotestosterone (DHT), a more than 1000-fold higher expression of transgene was observed compared to androgen negative control. In contrast to very high expression in the LNCaP cell line, the PSAR-PCPSA-P vector demonstrated minimal expression in non-PSA-producing prostate cell line PC-3, renal tumor cell line R11, breast cancer cell line MCF-7 and cervical adenocarcinoma cell line HeLa.
The high tissue specificity and androgen responsiveness of the combined construct, which consists of the newly identified DNA sequence and PCPSA-P, provide a solid foundation to generate gene therapy vectors for prostate cancer gene therapy.